Received: 30 May 2022 Revised: 23 December 2022 Accepted: 6 March 2023

DOI: 10.1111/1750-3841.16553

ORIGINAL ARTICLE
Fo o d E n g i n e e r i n g , M a t e r i a l s S c i e n c e , a n d Na n o t e c h n o l o g y

The fabrication of films based on polymer and containing

nanoclay, sodium diacetate, and tert-butyl hydroquinone for
packaging rainbow trout

Roonak Amiri1 Monireh Jahantigh2 Sahar Afsari Sardari1

Davoud Nassiri3

1 Department of Chemical Engineering,

Faculty of Engineering, Razi University,
Kermanshah, Iran
2 Department of Microbiology, Faculty of
Veterinary Medicine, Urmia University,
Urmia, Iran
3 Department of Food Hygiene, Naghadeh
Branch, Islamic Azad University,
Naghadeh, Iran
Correspondence
Davoud Nassiri, Department of Food
Hygiene, Naghadeh Branch, Islamic Azad
University, Naghadeh, Iran.
Email: davodnasiri2020@gmail.com
Funding information
Razi University
Abstract: This study investigates the fabrication of films based on a polymer con-
taining nanoclay, sodium diacetate (SDA), and tert-butyl hydroquinone (TBHQ)
for packaging rainbow trout fillets. The films were prepared by the addition of
2% SDA (SDA film), 2% TBHQ (TBHQ film), and a combination of both (1%
SDA + 1% TBHQ) into polyethylene polymer (93.00%) and montmorillonite
nanoclay (5.00%). A film lack of nanoclay, SDA, and TBHQ was prepared and
considered a control film. A film was also prepared by the addition of 95 g
polyethylene + 5 g nanoclay (Nanoclay). Morphological properties of the films
were investigated by a scanning electron microscope (SEM). In vitro antioxidant
properties and antibacterial activities of the films and their effects as the coat-
ing on fish samples were evaluated against Listeria monocytogenes, Salmonella
typhimurium, and Escherichia coli. The effects of films on oxidative stability,
antibacterial activity, pH, total volatile basic nitrogen (TVBN), and total viable
count (TVC) of fish samples were assessed. The SEM results showed the homoge-
nous dispersion of SDA and TBHQ into films. The SDA, TBHQ, and ST films
showed antibacterial activity against L. monocytogenes, S. typhimurium, and E.
coli compared with the control film as the coating and under in vitro conditions
(p < 0.05). The TBHQ and ST films exhibited higher antioxidant activity and pre-
vented the oxidation as the coating. The films prepared from the SDA, TBHQ, and
ST prevented an increase in TVC and TVBN (p < 0.05). The ST films can prevent
spoilage in fish samples and can be utilized in the food industry.

KEYWORDS
antibacterial, antioxidant, nanoclay, rainbow trout, sodium diacetate, tert-butyl hydroquinone

Practical Application: We successfully prepared films with the help of

nanoclay, sodium diacetate (SDA), and tert-butyl hydroquinone (TBHQ) on
polyethylene for packaging fish fillets. Films containing SDA, TBHQ, and nan-
oclay showed antibacterial activity and prevented spoilage. The films can be used
for packaging fish fillets.

2090 © 2023 Institute of Food Technologists. wileyonlinelibrary.com/journal/jfds J. Food Sci. 2023;88:2090–2103.


FILMS FOR PACKAGING RAINBOW TROUT
1 INTRODUCTION
Food spoilage commonly occurs during storage due to
the lipid deterioration of food products (Jacobsen et al.,
2019). Microbial spoilage is a result of chemical reactions
of products and causes economic losses (Odeyemi et al.,
2020). Fish is safe food, but it is sensitive to bacterial and
oxidant spoilage (Sheng & Wang, 2021). Rainbow trout
(Oncorhynchus mykiss) is a valuable fish species used in
both fresh and frozen forms (Ucak et al., 2020). It is a fatty
fish species vulnerable to microbial spoilage and deteriora-
tion (Rastiani et al., 2019). Preservation techniques should
be considered to maintain the quality of the product and
to improve extended shelf life (Domínguez et al., 2018).
Antibacterial and antioxidant films have been commonly
utilized to maintain food quality and extend shelf life.
Polymeric composites are containing inorganic/organic
fillers in form of flakes, spheres, and particulates (Idumah,
Hassan, et al., 2019; Idumah, Zurina, et al., 2019; Idumah
et al., 2020). Some composites have advantages such as
resistance to food spoilage and flexibility (Arfat et al.,
2018; Ariffin et al., 2018). Polyethylene is a polymer used
for food packaging with some properties, such as resis-
tance to solvents, water vapor barrier, and higher tensile
strength (Manikantan & Varadharaju, 2011). To improve
the mechanical and barrier properties of nanocompos-
ites, nanoparticles are utilized in nanocomposite materials
(Oliveira et al., 2021; Youssef & El-Sayed, 2018). Nanoma-
terials are used for food packaging with sizes of less than
100 nm (Idumah, Hassan, et al., 2019; Idumah, Zurina,
et al., 2019).
Other materials have been extensively applied into poly-
meric films. Nanoclays are clay crystallites connected by
stacking their layers (Guo et al., 2018). Montmorillonite
(MMT) nanoclay is consisted of thick aluminosilicate lay-
ers surface-substituted with metal and has 10 µm-sized
multilayer stacks (Taghvaei et al., 2021). The stacks are
scattered in a polymer matrix as fillers/additives and
increase mechanical strength, flame resistance, and thick-
ness (Irshidat & Al-Saleh, 2018). MMT nanoclays have
cation exchange capacity and could be combined with
dissimilar substances (Chanra et al., 2019), such as antibac-
terial and antioxidant compounds. Therefore, MMT can
improve the mechanical properties of polyethylene and is
combined with antibacterial and antioxidant compounds.
Tert-butyl hydroquinone (TBHQ) is an antioxidant com-
pound used in the food products, such as oils (Pu et al.,
2018). It was reported that adding 200 ppm TBHQ to
sesame oil hinders oxidative deterioration and improves
thermal stability (Prasad et al., 2015). Sodium diacetate
(SDA) is a sodium acetate salt produced by sodium car-
bonate and acetic acid or sodium acetate and acetic acid
(Ekhtelat et al., 2019) that is utilized as a fungicide and bac-
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2091
tericide in the food industry (Mohammadzadeh-Aghdash
et al., 2018). It is also used in food products as a safe
compound (Liu et al., 2017).
In the food industry, thermal-resistant antibacterial
and antioxidant films are required. Based on the liter-
ature review, several studies have prepared films based
on polyethylene-containing nanoclay for the packaging
of other food products (Bumbudsanpharoke et al., 2017;
Sabetzadeh et al., 2016). However, studies have not fab-
ricated polyethylene–nanoclay films containing SDA and
TBHQ for the packaging of fish samples. In the cur-
rent study, thermal-resistant antibacterial and antioxidant
films were produced using polyethylene as base and nan-
oclay for improving mechanical properties and being com-
bined with antibacterial and antioxidant compounds of
SDA, TBHQ, and their combination for packaging rainbow
trout.
2 MATERIALS AND METHODS
2.1 Materials
SDA (PubChem CID: 15820984) was prepared by Merck
Company with a melting point of 324◦ C. TBHQ (PubChem
CID: 16043) was purchased from Tokyo Kasei Kogyo Com-
pany with a melting point of 129◦ C. Low-density polyethy-
lene LF0200 was provided from Kosar Shimi Company
(Tehran, Iran) with MFI (230◦ C/2.16 kg) = 3.5 g/10 min and
density = 0.905 g/cm3 .
2.2 Methods
2.2.1 The preparation of the films
As nanoclay is a polar substance and polyethylene is
a nonpolar substance, nanoclay is not well distributed
into polyethylene. The modified MMT nanoclay was
purchased from Laviosa Chimica Mineraria Company
(Livorno, Italy). It was nanoclay modified with a qua-
ternary ammonium salt (dimethyl dehydrogenated tallow
ammonium) with safety approved for humans as reported
by previous studies (Gutiérrez et al., 2017; Kozak & Domka,
2004) and with a cation exchange capacity of 105 meq/100 g
clay. To prepare the films, different concentrations of nan-
oclay, polyethylene, SDA, and TBHQ were prepared and
investigated their morphological properties and antibac-
terial activities. The best responses were obtained in
the concentrations of 1% and 2% SDA and TBHQ. Five
films were produced as follows: (I) SDA film: 93 g
polyethylene + 5 g nanoclay + 2 g SDA, (II) TBHQ film: 93 g
polyethylene + 5 g nanoclay + 2 g TBHQ, and (III) ST film:

2092
93 g polyethylene + 5 g nanoclay + 1 g SDA + 1 g TBHQ.
A film was also prepared by the addition of 95 g polyethy-
lene + 5 g nanoclay (Nanoclay film). A film was also
prepared from pure polyethylene and considered control.
The mixtures were loaded into a twin extruder (Pishgaman
Sanat Company, Tehran city, Iran) by a 1-step blending and
melt-compounding. Conditions in the extruder included
processing temperatures of 180–200◦ C and screw speeds of
100–160 rpm. The prepared pellets were dried at 90◦ C for
24 h in a vacuum oven for removing the absorbed water.
2.2.2 Physical properties of films
The melting temperature, crystallization temperature, and
crystallinity percentage of the films were investigated
by a differential scanning calorimeter (DSC; TA Q100
MDSC) as reported by previous studies (Khalaj et al., 2016).
Tensile properties and barrier properties such as oxygen
and water vapor permeability were measured based on
recommendations of previous studies (Khalaj et al., 2016).
2.2.3 SEM analyses
The surface morphology of the film samples was investi-
gated by a scanning electron microscope (SEM; Hitachi
S-4300, Hitachi, Japan). Images were taken using an
accelerating voltage of 5 kV.
2.2.4 Microbial analyses
A panel of microorganisms, including Listeria monocyto-
genes (ATCC 19118) as Gram-positive bacteria as well as
Salmonella typhimurium (ATCC 14028) and Escherichia
coli O157:H7 (ATCC 10536) as Gram-negative bacteria,
was prepared from the culture collection of the Ira-
nian Research Organization for Science and Technology
(Tehran, Iran). Antibacterial activity of the bacterial sus-
pension containing approximately 1.5 × 108 CFU/mL was
assessed by the disc diffusion method as reported by oth-
ers (Ahmed et al., 2017). The incubation condition was
37 ± 1◦ C for 24 h, and the diameter of the incubation zones
was reported in mm. It is essential to mention that films
were cut into a disc shape by a metal hole puncher, and
both sides of the discs were sterilized with the help of
ultraviolet light for 15 min prior to testing.
Minimum inhibitory and bactericidal concentrations
(MICs and MBCs) were investigated by microtitre plates
by micro-dilution method as reported by previous stud-
ies (Rostami et al., 2012). The bacterial suspension
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FILMS FOR PACKAGING RAINBOW TROUT
was adjusted by sterile saline for a concentration of
1.0 × 105 CFU/mL and prepared dilutions in concentra-
tions between 0.078 and 20 mg/mL. The 96-well sterile
micro-dilution plates with U-bottom wells were prepared.
The microplates were incubated for 72 h at 28◦ C. The
lowest concentrations without visible growth were consid-
ered MIC. MBC was considered the lowest concentration
with no visible growth and/or with 99.5% killing of the
original inoculum (Farahpour et al., 2020). The optical
density of each well was considered at a wavelength of
655 nm by Microplate Manager 4.0 (Bio-Rad Laborato-
ries, Hercules, CA, USA) and compared with a blank and
the positive control. Streptomycin was used as a positive
control. Experiments were conducted in triplicate.
2.2.5 Antioxidant activity of the prepared
films
DPPH radical scavenging activities of films were investi-
gated as reported by previous studies (Tornuk et al., 2018).
In summary, the film samples of 0.1 g were singly cut into
small pieces and incorporated with 2 mL of methanol in a
test tube and kept at room temperature after being vortexed
for 3 min. It was repeated and centrifuged at 2300 rpm for
10 min. The supernatants were incorporated with 2 mL of
0.06 mM DPPH solution (in methanol). The absorbance
was investigated at 517 nm by a spectrophotometer.
2.2.6 The preparation of fish samples
Farmed rainbow trout (O. mykiss) slices were purchased
from a local market in Kermanshah City. Fish were cut
into small fillets (45 g each, 5 × 2 × 3 cm3 ), and the upper
and lower surfaces of the fillets were coated with the films
(7 cm × 7 cm) as reported by previous studies (Chang
et al., 2021). All the samples were kept at 25◦ C for 96 h and
investigated at time intervals of 12, 24, 48, and 96 h.
2.2.7 The investigation of total volatile basic
nitrogen (TVBN)
Total volatile basic nitrogen (TVBN) was measured with
the help of Kjeldahl steam distillation based on other stud-
ies (Amegovu et al., 2021). In summary, samples of 10 g
were homogenized, extracted with the help of Kjeldahl
steam distillation with 30% (w/v) aqueous sodium hydrox-
ide solution and 4% (v/v) aqueous boric acid, titrated
against 0.01 N hydrochloric acid, and the values were
reported.

FILMS FOR PACKAGING RAINBOW TROUT
2.2.8 The investigation of total viable count
(TVC)
Total viable count (TVC) was measured as reported by pre-
vious studies (Khoshnoudi-Nia et al., 2018). In summary,
10 g of each fish sample was first finely chopped, trans-
ferred into 90 mL sterilized 0.90% saline solution, and
homogenized. Then, samples of 1 mL of each dilution were
mixed with plate count agar (Beijing Aoboxing Bio-Tech
Co., Ltd., Beijing, China) in Petri dishes, incubated at 37◦ C
for 48 h, and enumerated.
2.2.9 Oxidative stability of the fillets coated
with the films
Thiobarbituric acid reactive substances (TBARSs) were
assessed as reported by previous studies (Khoshnoudi-Nia
& Moosavi-Nasab, 2019). In summary, the samples were
homogenized, rehydrated with distilled water, mixed with
trichloroacetic acid, and filtered. The filtrates and aqueous
thiobarbituric acid were mixed and incubated and their
absorption at 532 nm in a spectrophotometer was assessed
and reported.
The peroxide value (POV) of fish fillets was evaluated
as reported by other studies (Khan et al., 2015). In sum-
mary, samples were heated in a water bath for 3 min
at 60◦ C to dissolve the fat and homogenize the sample
after storage. Acetic acid–chloroform solution (3:2 v/v) was
used to dissolve the filets. The mixture was then filtered,
and saturated potassium iodide solution was added before
transferring into a burette and then titrated. The POV was
reported as milliequivalent peroxides per kg of the fish
sample.
2.2.10 pH value
To measure pH, samples were homogenized in 100 mL of
distilled water. The mixtures were filtered, and their pH
values were measured using a pH meter.
2.2.11 Antibacterial properties on fish
samples
Antibacterial activities of the films on fillets were assessed
based on previous studies (Woraprayote et al., 2018). In
summary, films were prepared and sterilized with the help
of UV for 15 min. Fillets (5 × 2 × 3 cm3 ) were inoculated
with the cocktail inoculum of E. coli, L. monocytogenes,
and S. typhimurium using adjustable volume micropipettes
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2093
on each side of separate fillets to a final concentration to
obtain the final bacterial density of each indicator on fish
surface 105 CFU/cm2 . The inoculated fillets were coated
with the films as mentioned previously and stored at
4 ± 2◦ C. Samples packed for 7 days and selective viable
counts of indicator strains were conducted. L. monocyto-
genes, E. coli, and S. typhimurium were enumerated on
PALCAM agar plates supplemented with PALCAM antimi-
crobic supplement, Eosin Methylene Blue Agar Modified
(Holt-Harris & Teague, Merk Company, Munich, Ger-
many) and XLD agar, respectively, after incubation, as
reported by previous studies (Woraprayote et al., 2018).
2.2.12 Statistical analysis
The analyses were conducted in triplicate and analyzed
for the effects of treatments. The data were investigated
for normality by Kolmogorov–Smirnov test, and signifi-
cant values were higher than 0.05; thus, the data were
normal. The data were analyzed by SPSS software (Ver-
sion 21, SPSS Inc, Chicago, IL, USA) and one-way ANOVA
analyses. The figures were illustrated by GraphPad Prism
Software (Version 6.07).
3 RESULTS AND DISCUSSION
3.1 The results of SEM
The micrographs in Figure 1 show a smooth surface lack of
any grain for nanoclay group (Figure 1). However, micro-
graphs showed that nanoclays, SDA, and TBHQ were well
distributed in the film matrix, and several fine agglom-
erations were seen in film matrix. The results highlight
that nanoclays, SDA, and TBHQ are well dispersed into
the film matrix, and the addition of SDA and TBHQ did
not have any significant effect on the formation of agglom-
eration. Fine agglomerations are due to the presence of
nanoclays in the film matrix. A concentration of 5.00%
of nanoclays was selected because concentrations higher
than 7.50% cause tactoids and clusters (Tornuk et al., 2018).
The results are in agreement with previous studies that
showed loading nanoclay into polymeric matrixes causes
fine agglomeration (Tornuk et al., 2018). In this study, a
constant amount of nanoclay was considered while others
believed that higher concentrations of nanoclays cause dif-
ficulty under different shear forces (Ataeefard & Moradian,
2011). In the current study, extrusion processing variables
were optimized to obtain a good polymer/filler interac-
tion because all the factors have significant effects on
improving interactions (Da Silva et al., 2010).

2094
FIGURE 1 The images for films based on polymer.
TA B L E 1 Differential scanning calorimetry results for the prepared films.
Films Melting T (◦ C)
Nanoclay 165.60
SDA 166.70
TBHQ 166.50
ST 166.10
Abbreviations: SDA, sodium diacetate; TBHQ, tert-butyl hydroquinone.
3.2 Physical properties of films
Table 1 depicts the results for the physical properties of the
prepared films. The results showed that melting tempera-
ture was lower in nanoclay films (165.60), whereas it was
raised with the addition of SDA (166.70), TBHQ (166.50),
and both (166.10). Crystallization temperature did not
show a significant difference with the addition of differ-
ent compounds. Crystallization percentage was decreased
with the addition of SDA (40.80%), TBHQ (40.60%), and
both (40.50%) compared with nanoclay (43.10). The results
for physical properties are in agreement with previous
studies (Khalaj et al., 2016). Seemingly, SDA and TBHQ
have impurities and influence physical properties.
The results of tensile properties and oxygen and water
permeability of the films are illustrated in Figure 2. The
addition of nanoclay into films significantly increased
tensile properties and decreased oxygen and water vapor
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FILMS FOR PACKAGING RAINBOW TROUT
Crystallization T (◦ C) Crystallization (%)
115.60 43.10
115.30 40.80
115.20 40.60
115.40 40.50
permeability compared with control films. However, the
addition of SDA and TBHQ could improve the mentioned
properties. The greatest effects could be attributed to
nanoclay. The results for the effects of nanoclay are in
agreement with previous studies (Khalaj et al., 2016). The
improved stiffness with the addition of other compounds
shows that MMT and other compounds are well dis-
tributed into the polymer matrix, which is in agreement
with SEM results. Seemingly, SDA and TBHQ form good
bands with MMT and improve tensile properties. With
regards to permeability, packages must produce barriers
for hindering the passage of oxygen and water vapor across
the polymeric matrix. These characteristics improve shelf
life and food organoleptic properties. The results for the
effects of nanoclay in improving permeability are in agree-
ment with previous studies (Dadfar et al., 2011; Osman
et al., 2007). Seemingly, MMT creates a physical barrier
for preventing oxygen absorption and decreases diffusion
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FILMS FOR PACKAGING RAINBOW TROUT 2095

F I G U R E 2 Mechanical properties of the prepared films for tensile properties (A) and oxygen permeability (B) and water permeability
(B). The letters show significant differences between groups. Letters (a–c) show significant differences among groups for each parameter. The
results were reported as mean ± SD.

F I G U R E 3 In vitro antibacterial activities of different films on inhibition zone (A), MIC and MBC (B). Letters (a–e) show significant
differences among groups foreach bacterium. The results were reported as mean ± SD.

by the film. Improved permeability in SDA and TBHQ
and/or ST could be attributed to tight bonds between clay
with compounds blocking water and oxygen permeability.
3.3 Antibacterial activity of the films
The results for in vitro antibacterial activity are shown in
Figures 3 and 4. The results for disc diffusion (Figure 3A)
showed that inhibition zones were significantly higher in
ST, SDA, TBHQ, nanoclay, and control films, respectively,
for all the bacteria. Any inhibition zone was not seen in the
control film. The values for MIC and MBC tests are shown
in micrographs 3B and 3C. The results showed that con-
trol films did not show any antibacterial activity even at
20 mg/mL. However, nanoclay film showed better antibac-
terial activity in MIC and MBC tests compared with control
films. The films prepared from SDA had greater bacte-
ricide and bacteriostatic activities. Concentrations used
for MIC and MBC were 2.50 and 5.00 mg/mL against
all the bacteria for SDA films. The films prepared from
TBHQ had lower antibacterial activities compared with
the SDA films. The ST films exhibited higher antibacterial
activity compared with other films (1.25 and 2.50 mg/mL

2096
F I G U R E 4 Diameter zone against different bacteria. Upper row = Escherichia coli, medium row = Listeria monocytogenes, low
row = Salmonella typhimurium.
for MIC and MBC tests). The ST films showed higher
antibacterial activities compared with nanoclay films. In
sum, control films did not show any antibacterial activ-
ity, whereas nanoclay showed better antibacterial activity
and SDA, and, especially, ST showed greater antibacterial
activity. Nanoclays are mostly utilized in the structure of
polymers for improving their physical characteristics in
the food industry (Bumbudsanpharoke & Ko, 2019). MMT
has received much attention in food packaging owing to
its high surface area with a large aspect ratio and good
compatibility (Farhoodi, 2016). The antibacterial activity
of MMT nanoclay is attributed to facilitate the exchange
of metal ions, such as Ag+ (Magana et al., 2008) and Cu2+
(Hu & Xia, 2006). Indeed, the surface chemistry of MMT
clays is changed by exchanging the predominant interlam-
inar cations with organic materials and inorganic cations
with a positive charge. However, nanoclay in combination
with SDA and TBHQ showed greater antibacterial activ-
ity that could be attributed to its mechanism. Nanoclay
releases active compounds for a longer time by enhanc-
ing their solubility in the nanocomposite and protects
foods for an extended period (Mirkhavar et al., 2018). The
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FILMS FOR PACKAGING RAINBOW TROUT
SDA showed a significant antibacterial activity that is in
agreement with previous studies (Lee et al., 2002; Qvist
et al., 1994). However, the mechanism of action for the
SDA is still unknown. The effects of SDA are attributed
to acetate groups of SDA that damage cellular membranes
(Mohammadzadeh-Aghdash et al., 2018). TBHQ showed
a medium antibacterial activity compared with SDA in
MIC and MBC tests. The antibacterial activity of TBHQ
might be attributed to the degradation and formation of
TBHQ, which is an intrinsic antibacterial compound (Ooi
et al., 2013). The results also showed that the ST films have
greatest antibacterial activity owing to synergistic interac-
tion effects between nanoclay, SDA, and TBHQ. Seemingly,
clay provides an appropriate bed for SDA and TBHQ as
antibacterial compounds.
3.4 In vitro antioxidant activity
The results of in vitro antioxidant activity of the films are
illustrated in Figure 5. Antioxidant properties were signifi-
cantly higher in TBHQ and ST films. The results did not

FILMS FOR PACKAGING RAINBOW TROUT
F I G U R E 5 Antioxidant activities of the prepared films as measured by DPPH radical scavenging ability (%). The results were reported as
mean ± SD. Letters (a–c) show significant differences among groups for same time.
show significant differences between SDA and nanoclay
with the control films. The ST films showed the high-
est antioxidant properties compared to other films at all
times. The results are in agreement with other studies on
the antioxidant activity of TBHQ (Karamac & Amarowicz,
1997) and SDA (Ma et al., 2020). TBHQ shows antioxidant
activity via scavenging free radicals. The results approve
that TBHQ and SA have synergistic interaction effects for
antioxidant properties under in vitro conditions. In the
current study, SDA did not show antioxidant activity in a
single form, whereas it showed its effects in combination
with TBHQ, which could be attributed to the poor antiox-
idant properties of SDA and its synergistic effects with
TBHQ. Nanoclay did not also exhibit antioxidant activity
which is in agreement with other studies (Javadifard et al.,
2022). In the food industries, antioxidants can decrease
lipid oxidation and improve food stability, which results in
the increased shelf life of the foods (McClements & Decker,
2018). Nanoclay did not have any antioxidant activity, but
it did not have also adverse effects on antioxidant activity
compared with the control group.
3.5 The results for TVBN and total
viable count
Figure 6A depicts the results for TVBN. The TVBN val-
ues were significantly higher in control and nanoclay films
at times of 12–96 h. The results showed that TVBN val-
ues increased with time passing. The highest values were
observed in the control–nanoclay, SDA–TBHQ, and ST
groups, respectively. The lowest values for TVBN were
observed in the ST group. The threshold limit of accept-
ability for the TVBN of rainbow trout is 20 mg N/100 g
(Khoshnoudi-Nia & Sedaghat, 2019; Lu et al., 2014).
In the current study, we observed values lower than
20 mg N/100 g in SDA, TBHQ, and ST films at all times.
However, the TVBN value was 20 mg N/100 g and higher
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2097
in control and nanoclay films at 48 and 96 h times. The
results show that loading SDA and TBHQ into films pre-
vented the increased TVBN in fish samples. Ammonia and
dimethylamine are considered TVBNs that are produced
as a result of destructive activities of microorganisms and
are the most important freshness indicators monitoring
the quality and safety of seafood products (Moosavi-Nasab
et al., 2021). In fact, TVBN is one of the main fish qual-
ity index markers, and an increase in TVBN value is due
to the activity of the microorganisms causing degrada-
tion and endogenous enzymes. Antibacterial activities of
the SDA, TBHQ, and ST films were previously discussed.
Sodium salts of low molecular weight organic acids, such
as acetic, lactic, and citric, have been used to prevent
microbial growth, increase sensory attributes, and improve
the shelf life of various foods. Seemingly, the treatment of
fish samples with the SDA, TBHQ, and ST films decreases
the activity of bacteria that could subsequently decrease
TVBN. Nanoclay did not have positive effects in decreas-
ing TVBN but it did not have also adverse effects on TVBN
compared with control group.
Figure 6B depicts the results for the TVC. The results
showed that TVC values were significantly higher in con-
trol and nanoclay groups at times 12–96 h. TVC values
increased with passing time. The highest values were
observed in the control and nanoclay groups. The lowest
values for TVC were observed in the SDA and ST films.
The ST and SDA groups did not show any significant differ-
ence. Studies have suggested 106 CFU/g for rainbow trout
(Arashisar et al., 2004; Javan et al., 2015). Our findings
showed that TVC was greater than 106 CFU/g in control
and nanoclay films at 48 and 96 h, whereas values were
lower than 106 CFU/g. The results show that films pre-
pared from SDA and TBHQ prevent an increase in TVC.
TVC is considered for studying the microbiological quality
of foods. The results for TVC confirm the results for micro-
biological analyses. TVC is usually used as the index for the
freshness of meat. The results for the effects of nanoclay
 17503841, 2023, 5, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.16553 by <Shibboleth>-member@ait.ie, Wiley Online Library on [22/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2098 FILMS FOR PACKAGING RAINBOW TROUT

F I G U R E 6 The TVBN (A) and totalviable count (logCFU/g) (B) for fish samples during 96 h. The results were reported as mean ± SD.
Letters (a–c) show significant differences among groups for same time.

F I G U R E 7 The effects of the treatments on TBARS (A) and POV (B) of fish samples during 96 h. The results were reported as mean ±
SD. Letters (a–d) show significant differences among groups for same time.

on TVC are in agreement with other studies (Javadifard
et al., 2022). Seemingly, TBHQ, SDA, and ST films have
antibacterial properties on a bed of nanoclay and decrease
bacterial count and subsequently TVC values. Nanoclay
did not have positive effects in decreasing TVC, but it did
not have also adverse effects on TVC compared with the
control group.
3.6 Oxidative stability
Figure 7A depicts the results for TBARS value. The results
showed that the TBARS values were significantly lower in
the ST and TBHQ films compared to other films (p < 0.05).
The control and nanoclay films did not show any sig-
nificant difference. Studies have reported TBARS values
<0.58, 0.58–1.51, and >1.51 mg/kg as not rancid, slightly
rancid, and rancid, respectively (Secci & Parisi, 2016). The
results showed that TBARS values were lower than 0.58
on 12 h for all groups. The results confirm non-rancid
at the same time. The values were 1.50 mg/kg for con-
trol, nanoclay, and SDA films on 24 h. It shows that
fish samples were slightly rancid, whereas any rancid-
ity was not seen for TBHQ and ST groups. The values
were higher than 1.56 for control, nanoclay, and SDA films
on 48 and 96 h, whereas values were by 1 for TBHQ
and ST groups at the same time. It shows that loading
TBHQ and its combination with SDA prevented rancidity.
TBARS are formed as a by-product of lipid oxidative dam-
age. In lipid peroxidation process, reactive oxygen species
and reactive nitrogen species attack carbon–carbon double
bonds in lipids. The process induces a mixture of complex
products, such as lipid peroxyl radicals, and hydroperox-
ides as the primary products, as well as malondialdehyde
and 4-hydroxynonenal as predominant secondary prod-
ucts (Tsikas, 2017). Nanoclay did not have positive effects in
decreasing TBARS but it did not have also positive effects
on TBARS compared with the control group. The results
for the effects of SDA are in agreement with other studies
on the effects of SDA on TBARS in sausage (Knight et al.,
2007; Sommers & Fan, 2003). The decrease in TBARS in
the TBHQ and ST films could be attributed to the antioxi-
dant activity of TBHQ as observed for in vitro antioxidant
activity. Decrease in TBARS in SDA, TBHQ, and ST films
might be attributed to their effects on the inhibition of
the formation of free radicals during the initiation step
and/or interruption of the propagation of the free radi-
cal chain reaction by acting as an electron donor and also

FILMS FOR PACKAGING RAINBOW TROUT
FIGURE 8 Antibacterial activities of different films on L. monocytogenes (A), S. Typhimurium (B) and E. coli (C) of fillet samples on
different days.
FIGURE 9 pH values of fillet samples at different times (h).
scavenging free radicals that are required to be evaluated
in future studies.
The results for POV in different groups are shown in
Figure 7B. The results are similar to the results obtained
for TBARS. The results showed that POV was significantly
lower in the ST, TBHQ, SDA, and nanoclay and/or control
films, respectively. The results did not show significant dif-
ferences between nanoclay and control films at all times.
The SDA films showed lower POV compared with control
and nanoclay films. The fillets coated with the SDA and
the TBHQ films had lower POV compared with other fil-
17503841, 2023, 5, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.16553 by <Shibboleth>-member@ait.ie, Wiley Online Library on [22/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
2099
lets coated by other films. It means that loading TBHQ into
the films reduces the POV and prevents oxidative in fillets.
Lipid oxidation is responsible for the quality degradation
of fillets during storage. POV is an important indicator to
measure the rancidity of fat. Fat oxidation leads to increase
in the formation of aldehydes and ketones and reduces the
quality of meat (Khan et al., 2015). Thus, TBHQ and ST
films prevent oxidation of the fillets compared with con-
trol films. The results for TBARS and POV are in agreement
with obtained results for DPPH values for the control, nan-
oclay, TBHQ, and ST films. The results for DPPH showed
that the DPPH value was low in control and nanoclay films,
and it was higher in TBHQ and ST films. There was only
a difference between SDA films in DPPH and their use as
a coating on fillets. The SDA films did not show antioxi-
dant activity in DPPH, whereas they showed antioxidant
activity compared with control films as the coating. The
difference between the DPPH tests with the tests on fillets
could be attributed to antibacterial activity that prevents
oxidation induced by bacteria as the coating.
3.7 Antibacterial activities against
bacteria on fillets
Figure 8 depicts the results for antibacterial activities of
the films on fillets on different days. The results showed

2100
that bacteria count was increased in the samples coated
with control films compared with other samples coated
with other films. The results showed that coating the fillets
with nanoclay could significantly prevent bacteria growth
compared with control films that are in agreement with
the obtained results for in vitro conditions. The results also
showed that ST, SDA, and TBHQ films had higher antibac-
terial activities, respectively. Based on findings, ST, SDA,
and TBHQ films reduced bacterial count compared with
the initial count of bacteria. It means that ST, SDA, and
TBHQ films have bactericide activities, whereas nanoclay
works as bacteriostatic. The results for antibacterial activi-
ties of ST, SDA, and TBHQ films on the fillets concur with
obtained results for in vitro antibacterial activities and also
obtained results for TBARS. In sum, ST films progressively
decreased total bacterial count during the first days. The
decrease observed during the first days is significant and
acceptable. It means that ST films have strong antibacterial
activity on fillets.
3.8 The results for pH value
Figure 9 depicts the results for pH values of the fish fillets
coated with the films. The results showed that pH values
were passing time in fillets coated with nanoclay and con-
trol films. The pH value was increased in other films after
24 h. Although the increased pH was 0.7 in the control
group, it had significant differences from other groups. The
increase in pH could be attributed to the increase in volatile
bases produced (Jouki et al., 2014), as shown for TVBN,
and it has negative effects on the quality of samples. The
results for pH are in agreement with the obtained results
for TVBN, as discussed.
4 CONCLUSION
In conclusion, we prepared films with the help of SDA and
TBHQ and a combination of both. In vitro studies and the
use of films as coating showed that the films have antibac-
terial activities, and greater antioxidant activity was also
seen in TBHQ and ST films. Films were used to coat fish
fillets and could prevent spoilage in fish fillets. The high-
est antibacterial and antioxidant activities under in vitro
conditions and on fillets were observed in films prepared
from both TBHQ and SDA films (ST films). TVBN and TVC
were two times lower and TBARS was by three times lower
times in ST films compared with nanoclay and control
films. In vitro antibacterial activity was almost five times
higher in ST films compared with nanoclay films. It can be
concluded that the addition of SDA and TBHQ has syner-
gistic effects for antibacterial activity and prevention of fish
spoilage. This preliminary study confirmed the efficiency
17503841, 2023, 5, Downloaded from https://ift.onlinelibrary.wiley.com/doi/10.1111/1750-3841.16553 by <Shibboleth>-member@ait.ie, Wiley Online Library on [22/05/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License
FILMS FOR PACKAGING RAINBOW TROUT
of the ST films in the preservation of rainbow trout sam-
ples. However, future studies with more tests are required
to investigate the effects of the ST films on fish samples.
AU T H O R CO N T R I B U T I O N S
Roonak Amiri: Conceptualization; Data curation; Formal
analysis; Funding acquisition; Investigation; Methodol-
ogy; Project administration; Resources; Software; Super-
vision; Validation; Visualization; Writing–original draft;
Writing–review & editing. Monireh Jahantigh: Formal
analysis; Validation. Sahar Afsari Sardari: Formal anal-
ysis; Validation. Davoud Nassiri: Funding acquisition;
Writing–original draft.
AC K N OW L E D G M E N T S
We appreciate Razi University for its support.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
We do not have any conflict of interest.
ORCID
Roonak Amiri https://orcid.org/0000-0003-0678-4080
Davoud Nassiri https://orcid.org/0000-0002-5626-3788
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How to cite this article: Amiri, R., Jahantigh, M.,
Sardari, S. A., & Nassiri, D. (2023). The fabrication
of films based on polymer and containing nanoclay,
sodium diacetate, and tert-butyl hydroquinone for
packaging rainbow trout. Journal of Food Science,
88, 2090–2103.
https://doi.org/10.1111/1750-3841.16553